生物技术通报 ›› 2022, Vol. 38 ›› Issue (5): 84-92.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1305
收稿日期:
2021-10-15
出版日期:
2022-05-26
发布日期:
2022-06-10
作者简介:
魏倩,女,硕士研究生;研究方向:昆虫分子生物学;E-mail: 基金资助:
WEI Qian1(), LIU Xiao-ning1(), ZHAO Jie2()
Received:
2021-10-15
Published:
2022-05-26
Online:
2022-06-10
摘要:
旨在研究2-十三烷酮胁迫对棉铃虫Helicoverpa armigera叉头框A类似蛋白(forkhead box A-like protein,FoxAl)基因表达水平的影响,及FoxAl蛋白是如何调控解毒酶基因CYP6B6表达,为进一步明确FoxAl参与棉铃虫解毒代谢和生长发育过程提供依据。通过酵母自激活检测FoxAl蛋白的转录激活能力;通过凝胶阻滞检测FoxAl蛋白与CYP6B6启动子的结合能力;通过RNAi沉默棉铃虫5龄幼虫中肠内FoxAl基因,检测不同时间(24,48,72和 96 h)后FoxAl和CYP6B6的表达变化情况;最后通过qPCR检测不同浓度(5,10和20 mg/g)2-十三烷酮处理不同时间(6,12,20,30和48 h)后棉铃虫6龄幼虫中肠内FoxAl和CYP6B6的表达谱,并分析FoxAl和CYP6B6表达谱的相关性。FoxAl蛋白具有激活酵母MEL1报告基因转录的能力,且该蛋白能与CYP6B6启动子中响应植物次生物质应答的核心片段结合。利用dsFoxAl沉默棉铃虫5龄幼虫中肠内FoxAl的表达量后,CYP6B6的表达量也显著降低。2-十三烷酮处理棉铃虫6龄幼虫后,中肠内FoxAl和CYP6B6的表达量变化情况相似,基本都呈抛物线趋势,在短时间12 h内两者的表达量迅速上升后,随胁迫时间的延长两者的表达量逐渐降低;此外,FoxAl和CYP6B6的表达量变化基本呈正相关,甚至在20 mg/g的胁迫浓度以及12 h和48 h的胁迫时间下两者都是高度正相关(r=0.819,P=0.045;r=0.987,P=0.007;r=0.978,P=0.011)。棉铃虫FoxAl蛋白可能是CYP6B6的转录激活因子,在植物次生物质2-十三烷酮短期胁迫下,FoxAl的表达量增加,进一步上调CYP6B6的表达,从而参与棉铃虫对2-十三烷酮的解毒作用。
魏倩, 刘小宁, 赵洁. 2-十三烷酮胁迫下棉铃虫FoxAl调控CYP6B6的表达[J]. 生物技术通报, 2022, 38(5): 84-92.
WEI Qian, LIU Xiao-ning, ZHAO Jie. FoxAl Regulating CYP6B6 Expression Under 2-tridecanone Stress in Helicoverpa armigera[J]. Biotechnology Bulletin, 2022, 38(5): 84-92.
图1 酵母Y2HGold(pGBKT7-FoxAl)菌株的转录激活功能检测 A:pGBKT7-FoxAl质粒的酶切鉴定(M:DL15000 DNA marker;1:pGBKT7-FoxAl质粒;2:pGBKT7-FoxAl质粒的EcoR I和BamH I酶切产物);B:Y2HGold(pGBKT7-FoxAl)菌株的菌落PCR鉴定(M:DL2000 DNA marker;-:阴性对照;1和2:两个转化后菌株);C:Y2HGold(pGBKT7-FoxAl)菌株在X-α-gal诱导下的自激活试验
Fig.1 Transcriptional activation test of transformed yeast Y2HGold(pGBKT7-FoxAl)strain A:Restriction digestion of pGBKT7-FoxAl plasmid(M:DL15000 DNA marker;1:pGBKT7-FoxAl plasmid;2:digestion products of pGBKT7-FoxAl plasmid by EcoRI and BamH I). B:Colony PCR identification of Y2HGold(pGBKT7-FoxAl)strain(M:DL2000 DNA marker;-:negative control;1 and 2:two transformed strains). C:Self-activation test of Y2HGold(pGBKT7-FoxAl)strain under the induction of X-α-gal
图2 棉铃虫FoxAl蛋白与CYP6B6启动子HE1片段的结合验证 A:凝胶迁移率检测FoxAl蛋白与HE1片段的相互作用 (泳道1: 1.0 ng HE1探针;泳道2: 1.0 ng HE1探针和1 μg FoxAl蛋白;泳道3: 1.0 ng HE1探针和1.5 μg FoxAl蛋白). B:凝胶阻滞试验证实FoxAl蛋白与HE1片段的结合 (泳道1: 1.0 ng HE1探针; 泳道2: 1.0 ng HE1探针和1 μg FoxAl蛋白; 泳道3: 1.0 ng HE1探针、1 μg FoxAl蛋白和标记探针100倍量的无关PGRP-B片段; 泳道4: 1.0 ng HE1探针、1 μg FoxAl蛋白和100倍量的未标记HE1片段)
Fig. 2 Verification of FoxAl in H. armigera binding to the HE1 fragment of CYP6B6 promotor A: Gel-shift assay of interaction between FoxAl and HE1 fragment (Lane 1: 1.0 ng HE1 probe; Lane 2: 1.0 ng HE1 probe and 1 μg FoxAl protein; Lane 3: 1.0 ng HE1 probe and 1.5 μg FoxAl protein). B: EMSA test of FoxAl combination with HE1 fragment (Lane 1: 1.0 ng HE1 probe; Lane 2: 1.0 ng HE1 probe and 1 μg FoxAl protein; Lane 3: 1.0 ng HE1 probe, 1 μg FoxAl protein and 100-fold labelled probe independent PGRP-B fragment; Lane 4: 1.0 ng HE1 probe, 1 μg FoxAl protein and 100-fold unlabelled HE1 fragment)
图3 FoxAl dsRNA 注射后棉铃虫5龄幼虫中肠内FoxAl和CYP6B6的相对表达量 在同一时间下用不同星号表示基因的相对表达量与对照组差异显著(*P<0.05,** P<0.01,*** P<0.001)
Fig. 3 Relative expression of FoxAl and CYP6B6 in the midgut of 5th instar larvae after FoxAl dsRNA injected Different asterisks indicate that the gene relative expression is of significant difference compared to control group under the same time(* P<0.05,** P<0.01,*** P<0.001)
2-TD处理2-TD treatment | r | P | |
---|---|---|---|
胁迫浓度 Stress concentration/(mg·g-1) | 5 | 0.532 | 0.178 |
10 | 0.653 | 0.116 | |
20 | 0.819 | 0.045 | |
胁迫时间 Stress time/h | 6 | 0.614 | 0.193 |
12 | 0.987 | 0.007 | |
20 | -0.326 | 0.337 | |
30 | 0.863 | 0.069 | |
48 | 0.978 | 0.011 |
表1 FoxAl和CYP6B6表达量的相关性分析
Table 1 Correlation analysis of FoxAl and CYP6B6 expre-ssion
2-TD处理2-TD treatment | r | P | |
---|---|---|---|
胁迫浓度 Stress concentration/(mg·g-1) | 5 | 0.532 | 0.178 |
10 | 0.653 | 0.116 | |
20 | 0.819 | 0.045 | |
胁迫时间 Stress time/h | 6 | 0.614 | 0.193 |
12 | 0.987 | 0.007 | |
20 | -0.326 | 0.337 | |
30 | 0.863 | 0.069 | |
48 | 0.978 | 0.011 |
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